This proposal seeks support to investigate in detail the covalent binding to liver protein of the products of metabolic activation of acetaminophen and isoniazid, two widely used therapeutic agents with established hepatotoxic properties. In addition, covalent binding of 3-hydroxyacetanilide, a non-hepatotoxic isomer of acetaminophen, will be studied. The specific objectives of the proposed investigation are: (1) to develop analytical methodology which may be used in the structural elucidation of drug-protein covalent adducts--high sensitivity mass spectrometric techniques, in combination with stable-isotope-labeling, will play a key role in this work; (2) to identify the nucleophilic functional groups on protein, and the electrophilic centers on drug metabolites, which participate in covalent bond formation; (3) to obtain indirect evidence for the structures of the reactive intermediates produced by metabolic activation of acetaminophen, 3-hydroxyacetanilide and isoniazid; and (4) to assess to what extent covalent binding of reactive electrophiles exhibits slectivity, both toward certain hepatic proteins and to specific amino acid residues within the protein structure. The experimental approach to be adopted in this investigation is novel in that endogenous protein, rather than exogenous neucleophiles, will be employed as "trapping agent" for reactive metabolites. The proposed studies should give basic information which will lead to a better understanding of the nature of drug-macromolecule covalent binding in vivo and in vitro and of those properties of xenobiotic small molecules which contribute to the development of drug-induced liver injury.